How is native mass spectrometry different and when would one apply it?

Native MS is a technique typically applied in protein analysis. The difference between “normal” and native mass spectrometry is in the conditions which are applied. Commonly electrospray ionization (ESI) of proteins is performed under acidic conditions, e.g. using diluted formic or acetic acid. Native mass spectrometry came up when scientists tried to analyse non-covalent protein complexes. These complexes are often unstable under acidic conditions and thus buffer conditions were changed to a more neutral pH range, typically around pH 6.5-8 using ammonia acetate or bicarbonate buffers.

Typical questions addressed by native mass spectrometry are:

  • how large is the complex under investigation
  • what are the constituents of the complex; is it homo- or heterogeneous?
  • Collision-induced dissociation experiments of the complex might also provide some structural insights e.g. about “core” components and components which are located at the surface of the complex

As an effect of the buffer changes the number of charges on the protein analytes usually drops, shifting the observed charge envelopes to higher m/z values.

Denaturated and native MS spectra of a monoclonal antibody
Denaturated and native MS spectra of a monoclonal antibody

What needs to be considered when using native MS?

Native MS allows for direct observation if intact multiprotein complexes up the the Megadalton mass range. Yet there are a few things to consider:

When doing native mass spectrometry, the pH is usually higher that with denaturated mass spec. As a consequence less protons are available for transferring the charge to the analytes. What we observe is a shift in m/z to higher values in comparsion to denaturated mass spectrometry as shown in the figure above. When analysing non-covalent complexes, a second effect observed: the m/z values of non-covalent complexes increases even faster with mass than that of globular protein. The reason is the loss of surface area. The charges are located at the surface of the analyte. Only a certain number of charges can be present on the surfaces as beyond a limit a charge repuslion will occur. When a non-covalent complex is formed, a part of the surface is involved in the inner interaction of the complex. That area cannot by used to localize charges. Therefore the mass-to-charge ration of non-covalent complexes is lower than the corresponding ratio of its constituents which is illustrated below:

Illustration of m/z shift by complex formation
Illustration of m/z shift by complex formation

As a consequence of this shift in m/z, the instruments hardware needs to be optimized. Quadrupoles need to be modified to be able to transfer these large ions. MS Vision is a leading company in modifying quadrupoles and and electronics for optimized transfer of high masses. Our current quadrupoles offer a quad mass value of 30-32.000 amu which allows to transfer ions approximately up to m/z 70-100.000 efficiently. In ongoing EU projects we are also developing new quadrupol technologies which will allow to go even higher.

Another aspect is the desolvatisation of the complexes. As they are ionized out of solution the protein complexes carry a lot of solvent molecules, typically water. To acquire good spectra it is necessary to get rid of those solvent molecules before the quadrupole (as when they are lost in the quadrupole mass filter, the mass/charge ration would change in the filter and transmission will be poor). For this, the pressure regime in the region between orifice and Q1 must be optimized in order to facilitate proper desolavtion. This can be achieved by pressure sleeves and/or needle valves to increase pressure and needs to be properly controlled.

It also important to note that quadrupole mass filters operate better at higher pressures as focussing of the ion beam is more efficient. This effect is shown on the relatively small PMMA polymer which shows much better sensitivity at elevated pressure in the Q1 region.

Improvement of ion transmission by pressure increase
Improved desolvation of proteins by applying higher collision energy

The collision energy in the systems front end also affects desolvation and thus the resolution. The figure shows the effect of solvent loss in after the ion source region. Resolution is poor and transmission for some charge states is not possible. By applying a higher collision energy the solvent molecules are stripped from the analytes and transmission can take place efficiently. MS Vision considers this in the high mass modifications by applying higher voltages for in-source collision to foster solvent stripping.

When native mass spectrometry shall be used a number of technical points need to be taken into consideration. MS Vision has a long-lasting experience in the design, manufacturing and on-site modification of Waters QTOF systems for dedicated high mass applications and is a trusted partner for leading scientific laboratories.

When you want to learn more about our offerings for native mass spectrometry, contact us at info@msvision.com or check out our native MS products!

Native mass spectrometry and non-covalent complexes – Literature:

Learn more about the application of MS Vision’s dedicated high mass QTOF and Synapt systems for native MS:

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